Dramatically improved RNA in situ hybridization signals using LNA-modified probes - PubMed (original) (raw)

Dramatically improved RNA in situ hybridization signals using LNA-modified probes

Rune Thomsen et al. RNA. 2005 Nov.

Abstract

In situ detection of RNA by hybridization with complementary probes is a powerful technique. Probe design is a critical parameter in successful target detection. We have evaluated the efficiency of fluorescent DNA oligonucleotides modified to contain locked nucleic acid (LNA) residues. This increases the thermal stability of hybrids formed with RNA. The LNA-based probes detect specific RNAs in fixed yeast cells with an efficiency far better than conventional DNA oligonucleotide probes of the same sequence. Using this probe design, we were also able to detect poly(A)(+) RNA accumulation within the nucleus/ nucleolus of wild-type cells. LNA-based probes should be readily applicable to a diverse array of cells and tissue samples.

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Figures

FIGURE 1.

FIGURE 1.

Short LNA-modified SSA4 RNA-FISH probes yield robust signals. (A) Schematic representation of the LNA/DNA probe design. The first, and fluorescently labeled, nucleotide of the probes is in all cases positioned at the SSA4 nucleotide immediately prior to the major poly(A)-tail addition-site [denoted p(A)]. A_n_ indicates the SSA4 RNA poly(A) tail. (B) SSA4 RNA-FISH analysis in the Δ_rip1_ mutant. Cells were grown at 25°C and fixed after a 20-min temperature shift to 42°C. This shift concomitantly induces SSA4 transcription and the Δ_rip1_-induced mRNA export phenotype. Fixed cells were analyzed by RNA-FISH using Cy3 5′-end-labeled LNA-modified or DNA probes (Fig. 1A; Table 1) and progressively more stringent hybridization conditions as indicated.

FIGURE 1.

FIGURE 1.

Short LNA-modified SSA4 RNA-FISH probes yield robust signals. (A) Schematic representation of the LNA/DNA probe design. The first, and fluorescently labeled, nucleotide of the probes is in all cases positioned at the SSA4 nucleotide immediately prior to the major poly(A)-tail addition-site [denoted p(A)]. A_n_ indicates the SSA4 RNA poly(A) tail. (B) SSA4 RNA-FISH analysis in the Δ_rip1_ mutant. Cells were grown at 25°C and fixed after a 20-min temperature shift to 42°C. This shift concomitantly induces SSA4 transcription and the Δ_rip1_-induced mRNA export phenotype. Fixed cells were analyzed by RNA-FISH using Cy3 5′-end-labeled LNA-modified or DNA probes (Fig. 1A; Table 1) and progressively more stringent hybridization conditions as indicated.

FIGURE 2.

FIGURE 2.

Poly(A)+ RNA-FISH analysis of the wild-type (WT), pap1-1 and Δ_trf4_ strains. Cells grown at 25°C or temperature-shifted for 30 min to 37°C were fixed and analyzed by RNA-FISH using a Cy3 5′-end-labeled LNA-modified dT20 probe (THJ790). For comparison, wild-type cells fixed after a temperature shift to 37°C for 30 min were also subjected to RNA-FISH using a dT70 DNA-oligonucleotide probe containing seven amino-modified thymidine residues for attachment of Cy3 fluorophores. To evaluate subnuclear localization, signals obtained using the THJ790 probe were overlaid with DAPI.

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